Evaluation of potential probiotic Lactobacillus casei strains by Kanokwan Tandee A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Food Science) at the UNIVERSITY OF WISCONSIN-MADISON 2013 Date of final oral examination: 12/21/12 The dissertation is approved by the following members of the Final Oral Committee: James L. Steele, Professor, Food Science Kirk L. Parkin, Professor, Food Science Amy C. Wong, Professor, Bacteriology Thomas D. Crenshaw, Professor, Animal Science Benjamin J. Darien, Associated Professor, Medical Science i Table of contents Abstract ii Acknowledgements iv List of figures v List of tables vii Chapter 1 Introduction 1 Chapter 2 Literature review 6 Chapter 3 Evaluation of potential probiotic Lactobacillus casei strains in 41 an in vitro gastrointestinal model and piglets Chapter 4 Transit of Lactobacillus casei 32G through the piglet ileum and 96 its effect on the composition of the ileum microbiota Chapter 5 Dose-dependent impact of Lactobacillus casei 32G on 141 the mouse cecum microbiota Chapter 6 Summary and recommendation for future studies 164 ii Evaluation of potential probiotic Lactobacillus casei strains Kanokwan Tandee Under the supervision of Professor James Steele At the University of Wisconsin-Madison Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Lactobacillus casei, due to its high level of consumption, is a probiotic species of particular interest. Significant genetic variability exists within this species with approximately 38% of the genes being variable between L. casei strains. This variability suggests that significant strain-to-strain variation in probiotic efficacy and effects is likely. This thesis describes methods for screening L. casei strains for attributes commonly associated with probiotics through the establishment of in vitro and in vivo models, and utilizes culture-independent methods to characterize the influence of consumption of L. casei on the composition of the gastrointestinal tract (GIT) microbiota of piglets and mice. Strain- specific differences were observed in the ability to survive gastric passage and adhere to the piglet ileum epithelial surface. These results led to L. casei 32G being selected for further characterization. The ability of L. casei 32G to alter the ileum digesta and epithelial tissue adherent microbiotas was examined in two separate piglet feeding trials. In both studies, significant changes were detected in the dominant genera in both the digesta and tissue samples; however, the specific genera that increased and decreased differed between the two iii trials. The second piglet trial also examined these alterations in the piglet ileum microbiota over time after the last dose. The results indicated that daily consumption of 32G resulted in significant, relatively short-lived alterations (hours) to the composition of both the digesta and Peyer’s patch microbiotas. The influence of 32G dose on the ability of this strain to alter the composition of the GIT microbiota was evaluated in mice. The results demonstrated that dose-dependent changes occur in the cecum microbiota of mice upon administration of L. casei 32G and that the lowest dose examined (106 CFU/day for seven days) had the most dramatic impact on this microbiota. In conclusion, L. casei 32G was selected as the strain examined with the greatest probiotic potential and was shown to cause restructuring of the GIT microbiotas in both the piglet and mouse models. iv Acknowledgements I am highly grateful for Dr. James Steele for his guidance and advice throughout my graduate study. I would like to thank my graduate committees - Dr. Kirk Parkin, Dr. Amy Wong, Dr. Thomas Crenshaw, and Dr. Benjamin Darien, as well as Dr. Jeff Broadbent and Dr. Nasia Safdar for their suggestions. I appreciate the technical assistance from Steele’s lab members - Dr. Vladimir Smeianov, Mateo Budinich, Dr. Hui Cai, Willyn Tan, Busra Aktas, Pamella Wipperfurth, Kari Nevermann, Dr. Ekkarat Phrommao, Kurt Selle, Lulu Hoza, Samuel Garber, Emily Engelhart, Jeehwan Oh, Elena Vinay-Lara, Jessie Heidenreich, Neil Gandhi, Davide Porcellato, Chokchai Chuea-nongthon, Pingfan Wu, Michael Donath, and Travis De Wolfe as well as students from University of Wisconsin-Madison Department of Animal Science, students from University of Wisconsin-River Falls Department of Animal and Food Science, Dr. Viriya Nitteranon, Simone Warrack, and Megan Duster. I finally thank Thai Government Ministry of Science and Technology for the scholarship as well as my family and friends for their encouragement and support. v List of figures Chapter 3 Figure 1 Survival of L. casei strains in the in vitro GI model 74 Figure 2 Detection of L. casei strains on the distal ileum-derived tissue 75 Figure 3 Discrimination between ileum digesta microbiotas of control and 76 32G fed piglets Figure 4 Discrimination between ileum tissue microbiotas of control and 77 32G fed piglets Figure S1 Detection of L. casei strains in the piglet intestinal digesta 83 Figure S2 Relative quantity of 16S-23S rRNA spacer amplicons found in the 84 control or 32G fed piglet digesta Figure S3 Discrimination between piglet ileum digesta and tissue microbiotas 85 Chapter 4 Figure 1 Transit of L. casei 32G in the piglet ileum digesta and Peyer’s patch 120 Figure 2 Clustering of the piglet ileum digesta microbiota at the genus level 121 by condition and sampling time after the last dose Figure 3 Clustering of the piglet ileum Peyer’s patch microbiota at the genus 122 level by condition and sampling time after the last dose Figure 4 Composition of piglet ileum digesta microbiota at the phylum or 123 genus level Figure 5 Composition of piglet ileum Peyer’s patch microbiota at the phylum 124 or genus level vi Figure S1 Discrimination between piglet ileum digesta and Peyer’s patch 130 microbiotas Chapter 5 Figure 1 Detection of Lactobacillus, coliform bacteria, and class Clostridia 160 in the mouse cecum Figure 2 Composition of mouse cecum microbiota characterized by ARISA 161 vii List of tables Chapter 3 Table 1 Origins and references for L. casei strains 78 Table 2 Log reductions of L. casei strains in the in vitro GI model and piglet 79 Table 3 Bacterial phyla, classes, orders, and families that differ between 80 control and 32G-fed piglets Table 4 Bacterial genera that differ between control and 32G fed piglets 82 Table S1 Humanized diet for 10 kg piglets per day 86 Table S2 Nutrient composition of the piglet humanized diet per day 87 Table S3 Log reductions of four L. casei strains in each compartment of 89 the in vitro GI model Table S4 Normalized weights by intestinal section of piglet digesta 90 Table S5 pH and lactic acid content of digesta from the last section of 91 small intestines of control or 32G fed piglets Table S6 ARISA profile of each piglet 92 Table S7 Bacterial genera detected in control or 32G fed piglets 93 Chapter 4 Table 1 Changes in composition of piglet ileum digesta microbiota resulting 125 from administration of L. casei 32G Table 2 Changes in composition of piglet ileum Peyer’s patch microbiota 127 resulting from administration of L. casei 32G. Table S1 Bacterial genera that differ between control and 32G-fed piglets 131 viii at 1.5 h after the last dose Table S2 Bacterial genera that differ between control and 32G-fed piglets 133 at 6 h after the last dose Table S3 Bacterial genera that differ between control and 32G-fed piglets 135 at 12 h after the last dose Table S4 Bacterial genera that differ between control and 32G-fed piglets 137 at 24 h after the last dose Table S5 Bacterial genera that differ between control and 32G-fed piglets 139 at 72 h after the last dose Chapter 5 Table 1 Average numbers of Lactobacillus, coliform bacteria, and Clostridia 162 in the mouse cecum Table 2 Identification of bacterial colonies isolated from Brucella agar plates 163 1 CHAPTER 1 Introduction 2 Our gastrointestinal tract (GIT) hosts over 1014 cells from at least 395 species of commensal microorganisms, which are 10 times the number of our own cells (5). The enormous number and complexity are an indication of the importance of our GIT microbiota. Additionally, the GIT microbiota is considered a human organ, as the changes in levels and compositions can influence metabolism and physiology involved in human health and disease (7). These microorganisms, mostly bacteria, have evolved in this complex and dynamic habitat and continued adapting as niches changed through a lifetime. Therefore, it is necessary to understand the role of the GIT microbiota to ensure the homeostasis that ultimately promotes our well-being. The GIT microbiota is destabilized by exogenous modifications, e.g. diet change, pathogenic infection, or antibiotic use, as well as endogenous factors, e.g. aging, obesity, or stress. Destabilization of the GIT microbiota can lead to imbalances in this microbiota, referred to as dysbiosis, which may ultimately result in disease (4). The ability of commensal bacteria to recover to a typical state is individual-specific. This is primarily due to genetic background, with factors such as metabolic rate and immune function thought to have key roles. Additionally, diet and exercise also play crucial role in controlling the GIT microbiota. Not surprisingly, health-promoting diets have drawn the public attention because of their impact on the digestive system and an indirect effect on the GIT microbiota. Furthermore, the concept of probiotics, which means “for life” in Greek, has emerged and gained popularity during the past century. This concept was first proposed by Elie Metchnikoff based upon his belief that consumption of beneficial bacteria in foods, such as yogurt, modified the intestinal flora and prevented illness, thereby enhancing longevity in Bulgarian peasants (1). 3 The Food and Agriculture Organization (FAO) and the World Health Organization (WHO), have defined probiotics as live microorganisms which when administered in adequate amounts confer a health benefit on the host (3).